Recombinant Adeno-Associated Virus (rAAV) vectors hold great promise for therapeutic treatment of a variety of inherited and acquired diseases. rAAV vectors have shown therapeutic efficacy in a variety of animal models and in clinical trials for gene delivery to various tissues such as liver, skeletal muscle, central nervous system, and the retina. rAAV vectors are attractive for gene therapy approaches since the vectors may be purified to high titers, used to infect dividing or non-dividing cells, and usually remain episomal in transduced cells greatly reducing the risk of insertional mutagenesis. Current approaches to produce rAAV vectors include transfection of producer cell lines and infection using adenovirus (Ad), herpesvirus, or baculovirus vectors. Each of these approaches is useful for rAAV production but each approach also suffers from one or more significant limitations for large-scale rAAV vector production. Thus, there is a significant need to develop an efficient, readily scalable, alternative approach to produce clinical grade rAAV vectors. rAAV vector production relies, in part, on the ability to express the AAV Rep and Cap proteins. Prior attempts to maintain and express the Rep gene in an Ad vector proved very difficult since the Rep gene was not stable due to an unknown toxicity to Ad replication. We used computational engineering to generate a Rep gene that is stably maintained over serial passage in a recombinant Ad vector. This observation will allow us to develop approaches to utilize Ad for the efficient production of rAAV vectors, a breakthrough in the field. The first aim of this proposal is to develop an Ad vector expresses the AAV2 Rep and Cap proteins in a temporally coordinated manner and at the appropriate expression levels to direct optimal rAAV production. The second aim of this proposal is to evaluate the ability to produce rAAV on a large-scale using Ad vectors and to compare the efficiency of the Ad-based system to rAAV production using the herpesvirus and baculovirus approaches. These approaches will provide the foundation for the efficient and large-scale production of rAAV vectors for use in gene therapy.